The molecular mechanisms that accomplish protein trafficking at the membrane of the endoplasmic reticulum (ER) are poorly understood, as are the molecules and mechanisms that mediate ER-associated degradation (ERAD), the movement of misfolded proteins from the ER lumen through the ER membrane to the cytosol ["retro-translocation" (RT) or "dislocation"] for degradation. Several unknown or unresolved fundamental issues essential for understanding the structure, function, and regulation of the molecules and assemblies involved in co-translational trafficking and RT will be addressed by the experiments proposed in this application. These issues include: How is the permeability barrier of the ER membrane maintained during the co-translational integration of multi-spanning membrane proteins (MSMPs) into the bilayer at the translocon? At what point does every other TMS in a MSMP rotate by 180o before moving laterally into the bilayer? Does each TMS in a MSMP fold into a 1-helix or nearly so inside the ribosomal nascent chain tunnel? Does TMS folding control translocon pore closure? Where and when do MSMP TMSs begin assembling into a native structure? Does an ERAD substrate move through the membrane in a folded or linear conformation? Which cellular proteins are required for the RT and degradation of a non-secreted Ig k light chain that contains disulfide bonds and is ubiquitinated? Does ricin enter the cytosol through the ER membrane, and if so, how? By site-specifically labeling the trafficking or RT substrate with a fluorescent or photoreactive probe, each process can be monitored from the perspective of the substrate. Probes are incorporated into substrates either chemically or during their synthesis by the inclusion of modified aminoacyl-tRNAs (aa-tRNAs) in an in vitro translation. In addition, two new unique approaches will be used. In one, the folding of ribosome-bound nascent chains is detected using fluorescence resonance energy transfer (FRET);in the other, the real-time kinetics of RT are monitored spectroscopically and continuously in biochemically well-defined mammalian in vitro samples. With these approaches, the environments and interactions of proteins undergoing co-translational trafficking or RT at the ER membrane can be characterized directly to determine what happens when protein trafficking systems work normally and also when processes such as protein folding go awry. Public Health Relevance: The experiments proposed in this application will examine, at the molecular level, the mechanism by which large protein molecules are moved through and inserted into a cellular membrane without disrupting its permeability barrier. Other experiments will characterize the folding of newly synthesized proteins, as well as the molecular interactions that mediate the degradation of proteins that fail to fold or assemble properly. Since an increasing number of diseases have been shown to be due to protein misfolding, such basic research is necessary to understand how proteins fold normally in the cell, and hence to identify by comparison how protein folding goes awry in abnormal or diseased cells.